Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C1/00—Magnetic separation
  • B03C1/02—Magnetic separation acting directly on the substance being separated
  • B03C1/28—Magnetic plugs and dipsticks
  • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03C2201/00—Details of magnetic or electrostatic separation
  • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications

Definitions

• the invention relates to apparatus for retaining magnetic particles in self- assembled magnetic particle structures in a liquid flow, and the use of such apparatus in particular in life science, chemistry and microfiltration applications.
• the invention concerns in particular an apparatus and a method wherein the magnetic particles are used for selectively capturing target molecules or target particles suspended in and carried by a fluid flowing through a flow-through cell, as is done for instance in clinical chemistry assays for medical diagnostic purposes.
• magnetic particles ('beads') embedded in a liquid can be used to carry a probe molecule on their surface that specifically interacts with a complementary target molecule (for example single stranded probe DNA interacting with complementary target DNA).
• a complementary target molecule for example single stranded probe DNA interacting with complementary target DNA.
• one can determine the amount of target molecules on a bead or within a certain volume containing beads see for example Hsueh et al., Techn. Digest Transducers'97, p. 175 (1997)).
• the very interesting point of using magnetic microbeads is that they can be manipulated using magnetic fields irrespective of fluid motion.
• beads have been locally fixed by using external magnets or have been transported using mechanically moving external magnets. The latter procedure may be for example used to fabricate mixing devices (Sugarman et al., US patent 5,222,808 (1992)) and in immuno-assay methods (Kamada et al., US patent 4,916,081 (1987)).
• An elegant way to keep magnetic particles in a fluidic channel was based on an electromagnet consisting of a coil and at least one pair of poles of a magnetic material.
• EP 1,331,035-Al Such poles form an inhomogeneous field transverse to the channel which effectively 'traps' the particles in regions where the field is strongest.
• EP 1,331,035-Al describes a flexible way of handling magnetic particles, though this requires a complex magnetic apparatus and electrical current manipulation.
• Specially corrugated pole tips need to be realised to generate the locally inhomogeneous magnetic field.
• Yellen and Friedman describe the use of micropatterned holes in a photoresist layer with at the bottom of the hole, a ferromagnetic thin film. When placed in a magnetic field, the magnetic film focuses the field and a magnetic chain is formed when a magnetic bead-containing solution is placed above the substrate. The idea was to form arrays of bead chains at regular positions of the substrate.
• Magnetic particle columns have thus been used in microfluidic channels, but in flow-through channels of homogeneous cross-section, so that the resistance to a fluid flow is minimum.
• the invention relates to an apparatus for retaining magnetic particles in self- assembled magnetic particle structures in a liquid flow, of the type comprising a flow- through cell or channel in which magnetic particles are suspendable in a liquid that is flowable through the cell or channel, and means for generating a substantially static magnetic field across the cell or channel such that when magnetic particles are suspended in a liquid in the cell or channel and the magnetic field is applied the particles form magnetic particle structures that are sustained by magnetic forces acting on the particles.
• the flow-though cell or channel has along its length transverse large sections alternating with narrow sections. The large sections are periodically distributed along and on either side of the narrow sections, arranged such that in use magnetic particle structures form across the large sections of the cell or channel.
• the liquid is flowable along the cell or channel through the narrow sections and through corresponding middle parts of the magnetic particle structures in the large sections, the magnetic particle structures being retained by engagement of end parts of the magnetic particle structures in the large sections of the cell or channel.
• the alternating large and narrow sections in the flow-though cell or channel permit the formation of chain- like magnetic particle structures using simple magnetic apparatus and good retention of these chain- like magnetic particle structures in the flow- though cell or channel without a need for additional retaining means.
• the invention also relates to a corresponding method for retaining magnetic particles in self-assembled magnetic particle structures in a liquid flow, in particular comprising flowing through the cell or channel a fluid carrying molecules or particles to be captured, filtered or activated by the magnetic structures, as well as uses of the apparatus and further features of the apparatus, as set out in the claims and the following description.
• Fig. Ia is a schematic plan view of an apparatus according to the invention
• Fig. Ib shows a detail of Fig. 1 on an enlarged scale, looking from above Fig. Ia
• Fig. Ic is a schematic end view of an apparatus according to the invention, looking along the x direction of Fig. Ia;
• Fig. Id shows a detail of Fig. Ic on an enlarged scale, looking sideways at the channel in the middle of the device, along the y direction of Fig. Ic;
• Fig. 2a is a schematic diagram showing magnetic beads in a microfluidic channel structure of varying width, with no liquid flow and no applied magnetic field;
• Fig. 2b is a corresponding diagram still with no liquid flow, but with an applied magnetic field;
• Fig. 2c is a corresponding diagram with liquid flow and with an applied magnetic field;
• Fig. 3 is a diagram of two interacting beads
• Figs. 4a and 4b are diagrams of two interacting magnetic chains; Figs. 5a, 5b and 5c show different ways of parallelising the implementation of the invention in microfluidic structures, and
• Figs 6a and 6b show a lay-out and a photograph of an experimental version of the invention.
• Fig. 1 schematically shows an apparatus according to the invention for retaining magnetic particles in self-assembled magnetic particle structures in a liquid flow, seen in plan view in Fig. Ia and end view in Fig. Ic.
• the apparatus comprises a microfluidic flow-through channel 5 in which in use magnetic particles are suspended in a liquid that is flowable through the channel 5.
• two permanent magnets, or electro- magnets, 1,2 are arranged for generating a substantially static magnetic field across the channel 5 such that when magnetic particles 12 (Fig. 2a) are suspended in a liquid in channel 5 and a magnetic field H is applied the particles form magnetic particle structures 15 (Fig. Ia; Fig. 2b) that are sustained by magnetic forces acting on the particles, as explained below in connection with Figs. 2a to 2c.
• the magnetic field H is typically comprised between 0.01 Tesla and 1 Tesla.
• the micro-channel 5 is contained in a microchip 4 that can be loosely placed (or fixed) in a recess 3 between the magnets 1,2.
• the microchip 4 is made of a plate of plastics material, or any other suitable non-magnetic material that has no magnetic shielding effect on the magnetic field, this plate having therein a central longitudinal channel 5 that has inlets and outlets 6,7 for connection to an external supply of liquid containing magnetic particles and other components, depending on the end use.
• the flow of liquid in the micro-channel 5 can be produced by a hydrodynamic or electrokinetic (electrophoretic and electro-osmotic) pumping mechanism, not shown.
• the apparatus is typically used with magnetic particles 12 in the range of the nanometer to a few micrometers.
• the size and the nature of the magnetic particles or beads 12 can of course vary for different applications.
• the chains 15 of magnetic particles are used to selectively capture target molecules, filter the liquid flowing in the micro-channel or catalyze chemical reactions at the surface of the magnetic chains.
• the micro-channel 5 has along its length transverse large sections 8 alternating with narrow sections 9.
• the large sections 8 are periodically distributed along and on either side of the narrow sections 9 and are arranged such that in use magnetic particle structures 15 form across the large sections 8 of channel 5, as shown in Fig. 2b.
• the liquid can flow along channel 5 through the narrow sections 9 and through corresponding middle parts of the magnetic particle structures 15 in the large sections 8 (Fig. 2c), the magnetic particle structures 15 being retained by engagement of end parts 18 of the magnetic particle structures in the large sections 8 of channel 5.
• W designates the spacing between the large sections 8, and the width of the large sections 8 along the direction of the channel 5 preferably corresponds to about W/3. These dimensions are typically of the order of a micrometer to a few tens of micrometers. The depth of the microchannel 5 is also typically one micrometer to a few tens of micrometers.
• the dimensions of the large and narrow sections 8,9 of the channel 5 can be changed as well as their periodicity.
• the transverse width of the narrow sections 9 is between 1 ⁇ m and 100 ⁇ m, and the transverse width of the large sections 8 is between l ⁇ m and 10 ⁇ m.
• the large section 8 of the micro-channel 5, when viewed from the top, can be rectangular, triangular or round tapered shapes.
• the invention implements a very simple solution for forming and retaining the magnetic particle structures 15, by providing a microfluidic channel 5 with varying cross section perpendicular to the flow.
• Fig. 2a shows such a channel with a smaller section 9 of size a and a larger section 8 of size b.
• a 30 ⁇ m
• b 60 ⁇ m.
• the depth of the channel 5, perpendicular to the plane of the drawing, can be a few micron, as is common in a microfluidic device.
• the micro-channel 5 can comprise sub-structures along the channel axis for enhanced retention of the magnetic chains 15, for example, micro-pillars (like the pillars shown in Fig. 4c) placed just downstream of the middle parts of the magnetic chains 15.
• the chains 15 of magnetic particles are formed at the position of the largest section b, for two reasons.
• a longer chain is characterised by a smaller magnetic demagnetisation factor in the direction of the field H and hence forms a magnetic object with lower magnetostatic energy than a shorter chain. This explains the situation of Fig. 2b.
• shear forces act to the chain 15 and a maximum shear force will occur at the point where the width of the channel 5 goes from a to b. If we magnify the two particles 12 at the lower part of the channel 5, we see that the maximum dipolar force is obtained when the angle ⁇ defined with respect to the field is 45°. This can be understood by analysis the forces taking effect on a magnetic chain.
• a magnetic chain that forms in the microfluidic channel structure consists of many individual particles.
• Figure 4 shows a chain formed by N particles (i ls ⁇ 2, .... , iisf) in the lower part and a chain formed by M particles (J 1 , J 2 , , JM) in the middle part of the channel structure.
• the magnetostatic interaction energy between both chains can be calculated as the summation of all mutual dipole-dipole interactions in the two-chain structure.
• the largest contribution to the interaction energy of both chains originates from the interaction between particle ii and J M , as their distance is shortest. Therefore, as a first approximation to the chain-chain magnetostatic energy, the magnetostatic interaction energy between these two particles as simply described by equation (1), can be considered.
• the magnetic interaction energy between two dipoles is given by:
• Fig. 3 is a schematic diagram of two beads 12 of a magnetic chain 15, where it can be seen that the lower particle 12 is held by the physical shape of the microchannel 5, while the top particle 12 is subjected to the flow.
• the tangential contribution to the total retained force is defined as:
• an array of cavities along the channel 5 will lead to the formation of a long-scale periodic structure 15 (made by many cylinders formed by the beads) inside the channel (Fig. 5a).
• an array made of parallel channels (Channel 1 and Channel 2) interconnected by cavities 8 will lead to a panel 17 of parallel structures (Fig 5b).
• the magnetic retention force within a channel 9 can be increased by adding pillar- like structures or posts 19 in the channel (Fig 5c). Each post 19 will create another holding spot for the chain, increasing the retention force by an amount 2 F ⁇ tot ⁇
• FIG. 6a shows a schematic diagram of the chip for retaining magnetic beads against a flow.
• Fig. 6b is a picture of an experimental array of magnetic chains contained inside the microchannel.
• Magnetic chains 15 by virtue of their high interaction with the liquid can capture or retain molecules, cells or particles carried by the liquid.
• the magnetic beads 12 can be coated with specific chemical groups, and have the role of probe for specific molecules.
• RNA molecules By choosing proper chemical groups (for example a well know single strand of a DNA or RNA molecule), one can capture the complementary strand. Therefore, due to the high specific interactions between the probe molecule on the bead and the target molecule carried by the flow, specific DNA or RNA molecules can be isolated from a complex mixture of molecules (a cell lyses solution for example). In a similar way, the magnetic chains 15 will capture target molecules from solution containing these molecules even in very low concentrations. One can later recover and concentrate the target molecules from the beads in a smaller volume. The molecules can then be used for analysis or PCR, for example.
• proper chemical groups for example a well know single strand of a DNA or RNA molecule
• Another promising application of the invention can be magnetic bead bio-sampling like use in a sandwich immunoassay.
• magnetic beads coated with specific antibodies would be used.
• a solution containing antigens will flow through the beads, and only the target antigens will be immobilized on the surface of the coated beads, while other antigen and undesired molecules will be washed out.
• the captured antigens can be released (and concentrated in a same way as above for the DNA or RNA), or labelled secondary antibodies can be flown over the beads and incubated with the immobilised antigens. Then, detection can be performed on-chip before washing the beads and starting another assay.
• the invention can be also used with any analytical procedure that requires interaction between antibody-coated beads and antigens. On-chip protein digestion can also be performed.
• Another interesting field of use of the invention can be in a microreactor. It is well known that a large amount of chemical reactions can take place in a microchip with a catalyst (atoms or small molecules) fixed on the walls of the microchip. The molecules used for catalysing the chemical reactions can be immobilised on magnetic beads. Due to the high interactions between the reagents and the catalyst on the bead (which leads to a complete reaction), its flexibility and the very small amount of reagents used (typically few hundreds of nanoliters to few tens of microliters), the invention can have an important impact in pharmacology research and development.

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• 2006
  • 2006-01-26 US US12/160,942 patent/US20100252507A1/en not_active Abandoned
  • 2006-01-26 EP EP06707847A patent/EP1981642A1/de not_active Withdrawn
  • 2006-01-26 WO PCT/EP2006/050459 patent/WO2007085300A1/en active Application Filing

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